To our knowledge, this is the first report of SMAD4 protein expression in early-onset colorectal cancer. We observed that 25% of colorectal cancers diagnosed before the age of 45 years lacked protein expression of SMAD4 and another 7% showed focal positive and negative staining suggesting clonal differences in SMAD4 expression. Therefore, the frequency of loss of SMAD4 protein expression in early-onset colorectal cancers falls within the broad range that studies of colorectal cancer of later onset have found for SMAD4 protein loss (~9–66%; 9.3% [5
], 14% [6
], 66% [7
]). This suggests that loss of SMAD4 expression in tumours is a common feature of all colorectal cancers irrespective of age at diagnosis.
A total of 45 of the 109 tumours (41%) analysed had at least one identifiable molecular disruption to the TGF-β signalling pathway. Twenty-seven tumours lacked the expression of SMAD4 and 18 of the SMAD4 protein-expression-positive tumours had frameshift mutations in TGFβR11. We also found that disruption of the TGF-β pathway is therefore involved in a large proportion of early-onset colorectal cancer.
We observed SMAD4 staining in the nucleus in both normal tissue and in positive-staining tumour tissue, indicative of a tumour suppressor gene active within the nucleus and that this nuclear staining was usually maintained even when TGFβ
R11 was lost because of a frameshift mutation. This observation is consistent with the findings of Montgomery et al.
, who demonstrated positive nuclear staining for SMAD4 in xenografted tumours from TGFβ
R11 negative SMAD4 +/+ cell lines [17
]. We did not observe an association between SMAD4 expression and TGFβ
R11 mutation status supporting other reports that these are independent molecular events that target the TGF-β
signalling pathway. We observed only three patients with early-onset colorectal cancer whose colorectal cancers lacked the expression of SMAD4 and also carried a frameshift mutation in TGFβ
R11 (and all three of these patients with early-onset colorectal cancer carried germ-line mutations in a MMR gene, see below). This suggests that any transforming or tumourigenic advantage conveyed by this dual hit is probably at best, small. TGFβ
R11 frameshift mutations have been measured as part of a 10-microsatellite marker panel to measure MSI in these early onset colorectal cancers [9
]. Similar to TGFβ
R11 status alone, we found no association between MSI status and SMAD4 expression (). This is consistent with other studies that have reported high rates of SMAD4 expression in MSI-positive tumours [7
These early-onset colorectal cancer patients have been screened previously for germ-line mutations in MMR genes [9
]. Of the tumours that lacked SMAD4 expression, three were MSI-high and all three patients with early-onset colorectal cancer carried germ-line MMR gene mutations (two in MSH2
and one in MLH1
). Of the tumours that expressed SMAD4, 14 were MSI-high and 10 of these carried germ-line mutations in a MMR gene (two in MSH2
and eight in MLH1
). Thirteen tumours were SMAD4 positive and MSI-low and four of these patients with early-onset colorectal cancer had germ-line mutations in MSH6
We also observed no association between SMAD4 expression status and SMAD4
copy number. Thirteen of the 27 (48%) of the tumours lacking SMAD4 expression showed some degree of genetic loss of SMAD4
(three had loss of both copies). This suggests there are other genetic or epigenetic events responsible for the loss of SMAD4 in tumours that have lost only one copy of the gene (). In addition, 14 tumours that were determined to have no change in copy number at the gene loci, despite an absence of staining for SMAD4, have possibly undergone two of these alternate molecular events. The work of others supports a role for somatic mutation of SMAD4
, which, while reported to be at high frequency in pancreatic cancers (20%) [18
], is relatively rare in colorectal cancer (3%) [9
]. A similar frequency of gene copy loss (14/29, 48%) was observed in tumours that expressed SMAD4. This could be because of a discrepancy in the IHC, but is most likely indicative of loss at 18q being frequent in colorectal cancer and that the retention of one copy is sufficient for normal protein expression, as measured by IHC (the biological implications of possible haploinsufficiency are unknown).
Loss of SMAD4 expression is a common feature of early-onset colorectal tumours as it is in colorectal cancer diagnosed in other age groups. The molecular mechanisms giving rise to loss of SMAD4 expression require further exploration as our data shows that it is not attributable exclusively to either SMAD4 copy number loss or MSI. Emerging and future genome-wide studies of somatic mutation, methylation and germ-line variation (GWASs) may give further insight into the possible mechanisms of loss of SMAD4 expression as well as identifying the molecular mechanisms underlying the majority of early-onset colorectal cancer.